Molecular imaging techniques have strong potential in the setting of preclinical research, primarily for drug discovery and basic science in the field of cancer research. In particular, high-frequency ultrasound is a powerful imaging modality for phenotyping and monitoring the response to experimental therapy. Relative to other molecular imaging techniques, ultrasound is portable, does not utilize ionizing radiation, is low cost, and has high spatial and temporal resolution. However, the development of efficacious molecular contrast agents that are active at the high imaging frequencies (15-40 MHz) used in small animal ultrasound imaging has greatly limited the application of this technique. Although ultrasound contrast agents for preclinical imaging are commercially available, the limited performance of these products has restricted the utility of this technique. Gas-encapsulated particles known as microbubbles (MB) are used as contrast agents for ultrasound, and recent research has identified MB diameter as the key determinant for MB activity as a function of imaging frequency. However, current MB formulations tend to be highly polydisperse, and there are currently no high- throughput techniques for manufacturing MB at controlled diameters suitable for commercial-scale manufacture. We propose to develop a low-cost method of producing MB of controlled diameter targeted to VEGFR2. Targeson has developed a lipid-encapsulated MB bearing a ligand against VEGFR2, and this agent has shown utility for imaging tumor angiogenesis at low ultrasound frequencies. We hypothesize that controlled dissolution of the lipid MB shell, by incubation in varying strength organic solvents, will enable us to reduce the mean diameter of this agent. Additionally, we have developed a robust method of gravitational separation that allows us to size-sort MB based upon differential buoyancy. We propose to combine these two methods to produce a series of VEGFR2-targeted MB populations of varying diameter. We will screen these MB preparations for imaging efficacy in vitro using a novel VEGFR2-coated ultrasound flow phantom and a high- frequency ultrasound scanner. We will then work with a commercial partner to test the ability of our size-sorted targeted MB to monitor tumor development in two mouse models of prostate and bladder cancer. PUBLIC HEALTH RELEVANCE: Molecular imaging can enable significant cost and time savings in basic science and drug discovery research. In particular, ultrasound molecular imaging has shown promise as a low-cost, high-throughput modality for imaging molecular markers of angiogenesis in a cancer setting. However, sensitive molecular contrast agents are not available for use at the high ultrasound imaging frequencies typically used for small animal cancer models. We propose to develop an ultrasound contrast agent that is precisely tuned to produce a reproducible and strong ultrasound contrast signal for use with small animal imaging in the preclinical research setting. Success in this project will enable routine use of ultrasound in the molecular imaging field and potentially increase the throughput and efficiency of cancer research.